The objective of this grant is to elucidate the regulation, downstream signaling, and structural dynamics of the glucan phosphatase laforin. Mammalian cells store readily mobilized energy in the form of glycogen, a water-soluble carbohydrate. Recessive mutations in genes encoding the E3 ubiquitin ligase malin or the dual specificity phosphatase laforin disrupt glycogen metabolism and result in a fatal, neurodegenerative epilepsy called Lafora disease (LD). A hallmark of LD is poorly branched, hyperphosphorylated insoluble carbohydrate accumulations called Lafora bodies (LBs), thought to be the causative agent of LD. We established that malin is a single-subunit E3 ubiquitin ligase that ubiquitinates and triggers the degradation of multiple proteins involved in glycogen metabolism. In addition, we discovered that laforin is the founding member of a unique class of phosphatases that dephosphorylate phospho-glucans. These results allowed us to propose molecular mechanisms that cause LD: 1) loss of malin results in LBs due to an imbalance in glycogen metabolism proteins, i.e. protein levels are not properly maintained; 2) loss of laforin results in LBs due to glucan hyperphosphorylation that inhibits glucan branching; 3) laforin also acts as a targeting subunit for malin, so that loss of laforin disrupts some malin-directed ubiquitination events. While we have made significant strides in determining the molecular mechanisms of LD, we lack an understanding of how these enzymes are regulated. This proposal will elucidate the deficiencies in our current knowledge. We recently identified novel phosphorylation and ubiquitination events on laforin. Phosphorylation and ubiquitination are post-translational modifications that direct changes in protein concentration, enzymatic activity, protein localization, protein-protein interactions, and structural dynamics. In Aim 1 we will define the in vivo conditions triggering laforin phosphorylation and characterize the functional consequences. We will utilize 1) overexpression and endogenous protein levels in tissue culture cells to monitor the status of laforin localization and modification, 2) assay laforin function in vitro using purified proteins, and 3) utilize a LD mouse model to verify our results. In Aim 2, we will utilize a similar strategy to determine the affects of ubiquitination on laforin function. In addition, we will define the role of laforin in malin directed ubiquitination, as we have recently discovered that laforin acts as a targeting protein for malin. In Aim 3, we will determine how perturbations of the structural components of glucan phosphatases contribute to LD. We will utilize Hydrogen- Deuterium exchange mass spectrometry to define the structural dynamics of laforin and x-ray crystallography to determine the structure of a glucan phosphatase. This proposal is built on our past discoveries and uses complementary approaches to advance our understanding of the intercalated events of cell metabolism, neurodegeneration, and epilepsy. Completion of this work will yield a better understanding of these complex events and will produce therapeutic insights for epilepsy and neurodegeneration. PUBLIC HEALTH RELEVANCE: The focus of our work is to determine how loss of function of either of two proteins leads to the fatal, neurodegenerative epilepsy called Lafora disease. Our proposal is built on our past discoveries and uses complementary approaches to advance our understanding of the intertwined events of cell metabolism, neurodegeneration, and epilepsy. Completion of this work will yield a better comprehension of these complex events and will produce therapeutic insights for epilepsy and neurodegeneration.